This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding enzymes involved in tryptophan biosynthesis in plants and seeds.
Many vertebrates, including man, lack the ability to manufacture a number of amino acids and therefore require these amino acids preformed in their diet. These are called essential amino acids. Plants are able to synthesize all twenty amino acids and serve as the ultimate source of the essential amino acids for humans and animals. Thus, the ability to manipulate the production and accumulation of the essential amino acids in plants is of considerable importance and value. Furthermore, the inability of animals to synthesize these amino acids provides a useful distinction between animal and plant cellular metabolism. This can be exploited for the discovery of herbicidal chemical compounds that target enzymes in the plant biosynthetic pathways of the essential amino acids and thus have low toxicity to animals.
Tryptophan is an essential amino acid. In plants, the biosynthesis of tryptophan from chorismic acid (see FIG. 1) requires five enzymatic steps catalyzed by anthranilate synthase (EC 4.1.3.27), anthranilate phosphoribosyl-transferase (EC 2.4.2.18), phosphoribosylanthranilate isomerase (EC 5.3.1.24), indole-3-glycerol phosphate synthase (EC 4.1.1.48) and tryptophan synthase (EC 4.2.1.20). The tryptophan pathway leads to the biosynthesis of many secondary metabolites including the hormone indole-3-acetic acid, antimicrobial phytoalexins, alkaloids and glucosinolates. Anthranilate phosphoribosyl-transferase is encoded by the PAT1 locus in Arabidopsis thaliana and the trpD locus in bacteria. Anthranilate phosphoribosyltransferase catalyzes the second step in tryptophan biosynthesis from chorismate forming 5-phosphoribosylanthranilate from anthranilate. Arabidopsis mutants in this gene are blue fluorescent under UV light due to accumulation of anthranilate compounds. Analysis of Arabidopsis plants expressing translational fusions of betaglucuronidase and different sections of the PAT1 gene indicates that the entire plastid transit peptide and the first two introns of PAT1 are required for efficient transcription and translation (Rose, A. B. and Last, R. L. (1997) Plant J 11:455-464). Anthranilate phosphoribosyltransferase purifies from Saccharomyces cerevisiae as a dimer (Hommel, U. et al. (1989) Eur JBiochem 180:33-40).
Phosphoribosylanthranilate isomerase catalyzes the third step in tryptophan biosynthesis from chorismate forming 1-(O-carboxyphenylamino)-1-deoxyribulose-5-phosphate. Three nonallelic genes encode phosphoribosylanthranilate isomerase in Arabidopsis thaliana. All three alleles contain a plastid transit peptide at their N-terminus, are over 90% identical and are flanked by nearly identical 350 nucleotide repeats (Li, J. Y. et al. (1995) Plant Cell 7:47-461).
Indole-3-glycerol phosphate synthase catalyzes the fifth step in tryptophan biosynthesis from chorismate producing indole-glycerol phosphate from 1-(2-carboxyphenylamino)-1-deoxyribulose 5xe2x80x2-phosphate. Mutation of seven invariant polar residues in the active site of the enzyme from Escherichia coli have allowed the identification of catalytically essential residues. Random saturation mutagenesis indicates that K114, E163, E53 and N184 are located in the active site of the enzyme (Darimont, B. et al. (1998) Protein Sci 7:1221-1232).
Few of the genes encoding enzymes from the tryptophan pathway in corn, soybeans, rice and wheat, have been isolated and sequenced. For example, no corn, soybean, rice or wheat genes have been reported for anthranilate phosphoribosyltransferase, phosphoribosylanthranilate isomerase or indole-3-glycerol phosphate synthase. Accordingly, the availability of nucleic acid sequences encoding all or a portion of these enzymes would facilitate studies to better understand cellular biosynthetic pathways, provide genetic tools for the manipulation of those pathways, provide a means to evaluate chemical compounds for their ability to inhibit the activity of enzymes in the tryptophan biosynthetic pathway.
The instant invention relates to isolated nucleic acid fragments encoding tryptophan biosynthetic enzymes. Specifically, this invention concerns an isolated nucleic acid fragment encoding an anthranilate phosphoribosiltransferase, an indole-3-glycerol phosphate synthase or a phosphoribosylanthranilate isomerase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding anthranilate phosphoribosiltransferase, indole-3-glycerol phosphate synthase or phosphoribosylanthranilate isomerase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a tryptophan biosynthetic enzyme selected from the group consisting of anthranilate phosphoribosiltransferase, indole-3-glycerol phosphate synthase and phosphoribosylanthranilate isomerase.
In another embodiment, the instant invention relates to a chimeric gene encoding an anthranilate phosphoribosiltransferase, an indole-3-glycerol phosphate synthase or a phosphoribosylanthranilate isomerase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding an anthranilate phosphoribosiltransferase, an indole-3-glycerol phosphate synthase or a phosphoribosylanthranilate isomerase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding an anthranilate phosphoribosiltransferase, an indole-3-glycerol phosphate synthase or a phosphoribosylanthranilate isomerase, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of an anthranilate phosphoribosiltransferase, an indole-3-glycerol phosphate synthase or a phosphoribosylanthranilate isomerase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an anthranilate phosphoribosil-transferase, an indole-3-glycerol phosphate synthase or a phosphoribosylanthranilate isomerase; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of anthranilate phosphoribosiltransferase, indole-3-glycerol phosphate synthase or phosphoribosylanthranilate isomerase in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding an anthranilate phosphoribosiltransferase, an indole-3-glycerol phosphate synthase or a phosphoribosylanthranilate isomerase.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of an anthranilate phosphoribosiltransferase, an indole-3-glycerol phosphate synthase or a phosphoribosylanthranilate isomerase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an anthranilate phosphoribosiltransferase, an indole-3-glycerol phosphate synthase or a phosphoribosylanthranilate isomerase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of anthranilate phosphoribosiltransferase, indole-3-glycerol phosphate synthase or phosphoribosylanthranilate isomerase in the transformed host cell; (c) optionally purifying the anthranilate phosphoribosiltransferase, the indole-3-glycerol phosphate synthase or the phosphoribosylanthranilate isomerase expressed by the transformed host cell; (d) treating the anthranilate phosphoribosiltransferase, the indole-3-glycerol phosphate synthase or the phosphoribosylanthranilate isomerase with a compound to be tested; and (e) comparing the activity of the anthranilate phosphoribosiltransferase, the indole-3-glycerol phosphate synthase or the phosphoribosylanthranilate isomerase that has been treated with a test compound to the activity of an untreated anthranilate phosphoribosiltransferase, indole-3-glycerol phosphate synthase or phosphoribosylanthranilate isomerase, thereby selecting compounds with potential for inhibitory activity.